We introduce the use of Arabidopsis thaliana callus culture as a system for proteomic analysis of plant organelles using liquid-grown callus. This callus is relatively homogeneous, reproducible and cytoplasmically rich, and provides organelles in sufficient quantities for proteomic studies. A database was generated of mitochondrial, endoplasmic reticulum (ER), Golgi/prevacuolar compartment and plasma membrane (PM) markers using two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (2-D SDS-PAGE) and peptide sequencing or mass spectrometric methods. The major callus membrane-associated proteins were characterised as being integral or peripheral by Triton X-114 phase partitioning. The database was used to define specific proteins at the Arabidopsis callus plasma membrane. This database of organelle proteins provides the basis for future characterisation of the expression and localisation of novel plant proteins.
We have identified a similarity between the apical domain of the human transferrin receptor and several other protein families. This domain is found associated with two different families of peptidases. Therefore, we term it the PA domain for protease-associated domain. The PA domain is found inserted within a loop of the peptidase domain of family M80M33 zinc peptidases. The PA domain is also found in a vacuolar sorting receptor and a ring finger protein of unknown function that may be a cell surface receptor. The PA domain may mediate substrate determination of peptidases or form protein-protein interactions.Keywords: aminopeptidase Y; cell wall-associated serine protease; transferrin receptor; vacuolar receptor Trafficking of soluble proteins through subcellular compartments is crucial to eukaryotic life. This process depends on integral membrane proteins that act as receptors. These bind lumenal proteins in one subcellular location. After transport of the receptor0lumenal protein complex to the target organelle the lumen proteins are released. While studying the structure of a plant specific vacuolar receptor, BP-80, we found distant homology in one domain to the mammalian transferrin receptor, involved in the endocytosis of transferrin. The occurrence of this domain in such diverse trafficking receptors was striking, and led us to further characterize the domain. The publication of the three-dimensional structure of the ectodomain of the transferrin receptor~Lawrence et al., 1999! allowed us to relate the sequence of these domains to a known structure.We used the common structural domain between BP-80 and the human transferrin receptor~residues 184-384 of P02786! as a query using PSI-BLAST~Altschul et al., 1997! with an E-value threshold of 0.01. The search converged after eight iterations. Using the BP-80 domain~residues 27-182 of P93484! as a query converges to essentially the same set of proteins. We have called this domain the PA domain for protease-associated domain. Based on the known structure of the transferrin receptor, the PA domain is 170-210 amino acids long and has a b-sandwich structure with two peripheral helices~see Fig. 1A!. The PSI-BLAST searches reveal that the PA domain occurs in four distinct families of proteins as described below.The plant vacuolar receptors homologous to BP-80~Kirsch et al., 1994; Paris et al., 1997! were found in the second round of searching with an E-value of 0.002. They have a large lumenal region that contains a PA domain at the N-terminus and three EGF domains preceding the C-terminal transmembrane helix and the short, tyrosine-targeting motif-containing, cytosolic region. These proteins bind the NPIR motif of soluble proteins destined for the lytic vacuole~Kirsch et al., 1994, 1996!. The protein is found in the prevacuolar compartments of plant cells~Sanderfoot et al., 1998!. There are at least 10 of these receptors in Arabidopsis thaliana, and they are conserved in both monocot and dicot plants, suggesting an important role for this family.We found th...
Quantitative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is used to determine changes in individual protein levels in complex protein mixtures. To provide reliable data, the software used for 2-D gel image analysis must provide a linear response over a wide dynamic range of data output. Here, we show that Phoretix 2D Full analysis of 2-D gels stained with colloidal Coomassie Brilliant Blue G-250 can provide a linear measure of changes in protein quantity. We show using a complex mixture of Arabidopsis thaliana proteins, that this is true for essentially all focused proteins, in a data output range greater than three orders of magnitude. An analysis of the factors that affect errors in the results demonstrated that reproducibility of the data is significantly improved by user seeding, whereas it is reduced by use of the background subtraction algorithms.
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